The invention relates to homocycsteinase enzymes that have a high level of specificity for homocycsteine as opposed to cycsteine. The invention also relates to enzyme preparations that comprise one or more homocysteinase enzymes and diagnostic kits containing these enzymes. Because of the high specificity of the homocycsteinase, hydrogen sulfide evolution can be used as a measure of the amount of homocysteine present. However, in addition, or in the alternative, the production of xcex1-ketobutyrate which is the product of homocysteine lysis can be used as a measure of the amount of homocysteine originally present.
As disclosed in PCT publication WO99/05311, published Feb. 4, 1999, and incorporated herein by reference, there is justified clinical interest in measuring the levels of blood plasma homocysteine due to the correlation of elevated plasma homocysteine concentrations and arteriosclerotic disease. Because of this interest and the diagnostic value of homocysteine levels as an indicator of cardiovascular condition and prognosis, improved methods for making this assessment are desirable. Further, our simplicity in such assays is a highly desirable characteristic. The present invention relates to an aspect of these assays in which resides the use of homocysteinases with improved specificity for homocysteine in comparison to cysteine.
xe2x80x9cHomocysteinasexe2x80x9d refers to a desulfurase that decomposes homocysteine to yield hydrogen sulfide, ammonia, and xcex1-ketobutyrate. Homocysteinases are generally not completely specific for homocysteine and also decompose cysteine to obtain hydrogen sulfide, ammonia, and xcex1-ketopropionate. In addition, normal levels of cysteine in blood plasma are considerably higher than those of homocysteine. The normal range of cysteine levels is about 30-120xcexc molar and those of homocysteine only about 5-15xcexc molar. Thus, if homocysteine content is to be measured by treating plasma with homocysteinase and measuring the common product hydrogen sulfide (or ammonia), the homocysteinase must be sufficiently specific for homocysteine in contrast to cysteine to provide an accurate measurement without interference from the cysteine levels. The present invention provides such homocysteinase enzymes.
In prior assays which have involved the use of homocysteinase and detection of hydrogen sulfide, such as that described by Thong, K. W., et al., Experimental Parasitology (1987) 63:143-151; Thong, K-W, et al., IRCS Medical Science (1985) 13:493-496, interference by the cysteine present was observed. In the case of the homocysteinases of the present invention, a sufficiently catalytic ability with respect to cysteine is achieved that an accurate measure of homocysteine in plasma can be directly obtained in a single enzyme assay using hydrogen sulfide as the detected product.
The present invention provides methods to determine the concentration of homocysteine in biological fluids, such as urine, tissue fluid, blood, blood serum, or blood plasma sample from a patient. These methods are useful to assess risk for cardiovascular disease. The methods determine homocysteine concentrations in biological fluids while avoiding detection of related but interfering substances, most particularly cysteine and methionine. The present invention achieves this by providing homocysteinase enzymes of sufficient specificity.
In one aspect, the invention is directed to a homocysteinase which is sufficiently specific for homocysteine as compared to its ability to decompose cysteine to provide a valid measure of homocysteine in a biological fluid even in the presence of normal amounts of cysteine. Preferably, the homocysteinase of the invention has the property such that at least about 90% of the hydrogen sulfide produced by action of said homocysteinase upon contacting a biological fluid is contributed by homocysteine when the concentrations of homocysteine and cysteine in the fluid are, respectively, about 5-15xcexc molar and about 100-300xcexc molar. Preferably, under these conditions, at least about 99% of the hydrogen sulfide produced by said homocysteinase is contributed by homocysteine. The invention also includes recombinant materials and methods to prepare the homocysteinase of the specificity required by the present invention.
Accordingly, there is provided a method for determining the amount of homocysteine that is present in a biological sample containing, for example, homocysteine and cysteine, that comprises contacting said sample with an enzyme preparation that produces hydrogen sulfide from homocysteine and determining the amount of homocysteine in said sample by measuring the amount of hydrogen sulfide produced from homocysteine. The enzyme preparation consists only of one or more enzymes whose reactivity toward homocysteine is sufficient, compared to cysteine, that hydrogen sulfide produced from cysteine may be ignored. Alternatively, xcex1-ketobutyrate may be measured as a correlate to homocysteine levels.
Said enzyme preparation comprises an enzyme referred to as a homocysteinase, and which may also be referred to by other names as described below. Additionally, the term xe2x80x9cenzyme preparationxe2x80x9d should also be understood to include, unless otherwise noted, both a single and a plurality of aliquots, that is, either a single amount or multiple amounts of one or more enzymes may be added during the course of an assay procedure, and the use of the term is without limitation has to how, or how many, aliquots or steps are used to add all of the necessary enzymes during an assay.
According to a preferred aspect of the invention, it is recognized that the total concentration of homocysteine present in biological samples, for example in body fluids, includes homocysteine molecules that are not present in free form, being instead covalently coupled to other molecules. The methods of the invention provide further steps for releasing this homocysteine prior to measuring of homocysteine-derived hydrogen sulfide.
It should be noted, however, that since the methodology of the present invention provides for accurate measurement of free homocysteine levels absent interference from related substances (for example, cysteine), valuable information is provided to the clinician even if only free homocysteine is detected. Among many uses, it is expected that such information is very useful as a fast initial diagnostic tool, for example, in the testing of all newborn infants.
In one preferred embodiment, the homocysteinase is a natively produced homocysteinase which has optionally been mutated to enhance its specificity. Techniques are described below whereby such mutations can be randomly generated and readily screened to select for those which result in homocysteinases of the required specificity.
In another preferred embodiment, the nucleotide sequence encoding the homocysteinase is derived from more than one gene or other polynucleotide that encodes a homocysteinase, wherein expression of such sequence leads to the production of a chimeric homocysteinase. A preferred example includes a chimeric enzyme that comprises amino acid sequences derived from both Trichomonas vaginalis and Pseudomonas putida homocysteinase.
Thus, the homocysteinase enzyme of the invention is sufficiently non-reactive toward cysteine or methionine that the concentration of homocysteine that is present, for example, in a sample of tissue fluid, urine, blood, blood serum, or blood plasma of a subject may be determined in a single step assay, wherein is measured the amount of one or more products resulting from reaction of said homocysteinase on homocysteine in said sample, and wherein said measurement is substantially unaffected by the concentration of cysteine or methionine therein.
In a particularly preferred embodiment the homocysteinase is a substitution variant, addition variant, deletion variant, or derivative of SEQ ID NO: 10, wherein said variant or derivative has one or both of the following properties:
(a) at least about 110% of the activity of SEQ ID NO: 10 toward homocysteine in a suitable assay; and/or
(b) no more than about 90% of the activity of SEQ ID NO: 10 toward cysteine (or methionine) in a suitable assay.
For the clinical practice of the invention, there is provided a diagnostic kit for use in determining the amount of homocysteine in a biological sample, and wherein the homocysteinase included therewith as described below. In a preferred embodiment, the diagnostic kit comprises (a) a homocysteinase of the required specificity, e.g., derived from that of Trichomonas vaginalis, or Pseudomonas (such as species ovalis or putida), and (b) at least one reagent capable of being used to determine the amount of hydrogen sulfide formed in the homocysteinase reaction.